An antibacterial agent produced by Staphylococcus epidermidis strain 5 was isolated from culture supernatants; its spectrum of activity is restricted to staphylococci and micrococci. The bacteriocin-like substance was purified to homogeneity by column chromatography on Servachrome XAD-2, CM-Sephadex C-25 and Sephadex G-50. Chemical analysis showed it to be a peptide with a molecular weight of about 6000 and an isoelectric point of about 10.5. Purified material was rapidly inactivated by trypsin, chymotrypsin and pronase and it was relatively heat-stable. After acidic hydrolysis 10 amino acid residues and two unidentified ninhydrin-positive substances were detected. Production of the bacteriocin-like substance was not inducible with mitomycin C.

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The staphylococcin-like peptide Pep-5 was shown to be a complex mixture of closely related and strongly basic peptides. Five peptides were purified by high-pressure liquid chromatography on reversed-phase and gel filtration columns and further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analysis. Four peptides have molecular weights of ca. 3,500, whereas one is of double size. All contain the thioether amino acid lanthionine and a large number of lysine residues per molecule. The amino terminus of the main active peptide is blocked; the carboxy-terminal end is formed by a lysine residue. The data obtained for Pep-5 suggest striking structural similarities to the peptide antibiotics nisin and subtilin.The staphylococcin-like peptide Pep-5 produced by Staphylococcus epidermidis 5 is a small and strongly basic peptide with a bactericidal action on members of the family Micrococcaceae (12). It has been shown to cause a rapid efflux of low-Mr compounds such as K+, amino acids, and ATP from the cytoplasmT of sensitive cells (14). Therefore, the cytoplasmic membrane appears to be the primary target of its action. As a consequence of the efflux of substrates, cofactors, and phosphorylated nucleosides, total inhibition of biosynthesis was observed after the addition of Pep-5 to growing cells (13). In early investigations on the structure of Pep-5 (12), a high content of hydrophobic amino acids was found in hydrolysates along with a large number of lysine and arginine residues, resulting in an isoelectric point of the peptide of about 10.5. Furthermore, two ninhydrin-positive substances were detected which could not be identified. These substances were supposed to contain the sulfur found in elementary analysis of Pep-5, which could not be attributed to the amino acids identified after acidic hydrolysis. These results prompted the more detailed investigations on the structure of Pep-5 reported in this paper. It is shown that Pep-5 can be fractionated into several closely related peptides with different specific activities. All peptides contain the rare amino acid lanthionine, which suggests similarities of Pep-5 to the known peptide antibiotics nisin and subtilin (3, 6). These are produced by group N streptococci and Bacillus subtilis, respectively, and strongly resemble Pep-S with respect to their molecular properties (9) and mode of action (11). MATERIALS AND METHODSStrains. The Pep-5-producing strain S. epidermidis 5 and the indicator strain S. cohnii 22 were maintained on blood agar plates and subcultured weekly. Production and purification of Pep-5 as well as the activity tests were performed as described previously (13).Analytical methods. (i) HPLC. For high-pressure liquid chromatography (HPLC) investigations a Gilson gradient system was used, including a Microsorb C-18 (particle size, 5 ,um; Rainin Instrument Co., Inc., Woburn, Mass.) or a Lichrosorb C-18 (particle size, 5 ,um; Bischoff Leonberg, * Corresponding author.Federal Republic of Germany) column. A precolumn (...

S U M M A R YTwo bacteriocins were found in the supernatant fluid and in an extract of Streptococcus faecium strain EI. The small soluble enterocin EIA represented more than go % of the total activity in the supernatant fluid, and was purified 400-fold by ammonium sulphate fractionation, gel filtration on Sephadex G-75 and chromatography on DEAE-cellulose. Enterocin EIB, with a particle weight greater than 4 x I O~, was the predominant type in the extract. It was released in appreciable quantities after breakage of the bacteria and was purified Ioo-fold by differential centrifugation, chromatography on Sepharose 4 B and density gradient ultracentrifugation. Enterocin EIA, a basic substance with a molecular weight of about 10000, was resistant to heat and was attacked by trypsin, whereas enterocin EIB was less thermostable and insensitive to proteolytic enzymes. The activity of enterocin EIB was unchanged by treatment with DNAase. Sensitivity to enterocin action was confined to certain strains of various enterococcus species, Streptococcus salivarius and Listeriu rnonocytogenes ; all the other Gram-positive and Gramnegative bacteria tested for sensitivity were unaffected by either enterocin.

Evidence for the presence of carbohydrate on the surface membrane of Toxoplasma gondii trophozoites and on the cell wall of toxoplasma brain cysts was sought by fluorescent lectin staining. Using FITC-conjugated preparations of Concanavalin A (Con A), wheat germ agglutinin (WGA), or soy bean agglutinin (SBA), we have failed to obtain evidence for the binding of these lectins on the surface of T. gondii trophozoites. In contrast, the three test lectins bound effectively and specifically to the wall of toxoplasma brain cysts. Prefixation of cysts with glutaraldehyde or brief trypsinization of cysts did not affect the intensity of cyst wall fluorescence when stained with FITC-conjugated Con A, SBA, or WGA. The results are interpreted to indicate that whereas exposed Con A, SBA, and WGA binding sites are associated with the wall of toxoplasma brain cysts, such lectin-binding saccharide residues are not present on the surface of trophozoites in exposed or reactive form.